Protocol biopsies in renal transplantation are those that are procured at predetermined times post renal transplantation, regardless of renal function. These biopsies have been useful to study the natural history of the transplanted kidney as they have detected unexpected - i.e. "subclinical" pathology. The most significant subclinical pathologies that have been detected with protocol biopsies have been acute lesions, such as cellular and antibody mediated rejection, and chronic lesions, such as interstitial fibrosis and tubular atrophy, and transplant glomerulopathy. The potential benefit of early recognition of the above lesions is that their early treatment may result in improved long-term outcomes. Conversely, the identification of normal histology on a protocol biopsy, may inform us about the safety of reduction in overall immunosuppression. Our centre, as well as others, is attempting to develop non-invasive methods of immune monitoring of renal transplant patients. However, we believe that until such methods have been developed and validated, the protocol biopsy will remain an indispensable tool for the complete care of renal transplant patients.

The decrease in the incidence of clinical re­jections and increased graft survival at one year has not translated into improved long-term out­comes in renal transplant patients. Current rates of graft loss are in the order of 2% per year.[1] The common histopathological features of the failing renal allograft include interstitial fibrosis and tubular atrophy (IF/TA), and varying deg­rees of arteriolar hyalinosis and glomeruloscle­rosis that are largely irreversible when detected in patients with graft dysfunction. The lesions are non-specific; however, they have been des­cribed in renal transplant patients after pro­longed exposure to calcineurin inhibitors (CNI), and, more recently, after polyoma virus infec­tion. As a result, there has been a proliferation of immunosuppressive drug regimens in which a reduction in immunosuppression is sought, and where attempts are made to avoid or mini­mize CNI; these regimens, however, require a careful assessment of their long-term safety, as the initial phases of allograft injury occur in the absence of allograft dysfunction.

Protocol biopsies have been useful for the de­tection of unexpected pathology in renal trans­plant patients with well-functioning grafts, e.g. subclinical acute rejection and subclinical peri­tubular capillaritis, and early chronic changes such as IF/TA and transplant glomerulopathy (TG). An emerging literature suggests that sub­clinical acute rejection and peritubular capilla­ritis, IF/TA and TG correlate with subsequent graft dysfunction and loss. The early recogni­tion of these histological changes may therefore result in optimization of the immunosuppres­sive regimen and in an improvement in both short and long-term patient and graft survival. Indeed, until non-invasive biomarkers for these pathologies are developed and validated, proto­col biopsies may be required for optimal patient care.

Subclinical Acute Rejection and Peritubular Capillaritis

Our group was the first to report that acute rejection - as defined in the Banff schema,[2] was present in up to one-third of well-functioning renal allografts in the first three post-transplant months, and the term "subclinical rejection" was coined.[3] However, an earlier paper had des­cribed lymphocytic infiltrates at one week and one month after transplantation in patients with good later outcomes.[4] Furthermore, at the Necker Hospital in Paris, Dr. Jean Crosnier described what he called "latent crisis", referring to histo­logical edema and lymphocytic infiltrates in 8 patients with normal graft function in whom "systematic biopsies" had been procured, and in whom the kidneys became less swollen after treatment with corticosteroids in 5 cases.[5]

Our definition of "subclinical" rejection was stringent, in that it required an increase in the serum creatinine of less than 10% from base­line, and a Banff score of "ai2at2" (Type IA acute rejection) or greater. The finding of sub­clinical rejection, as defined above, was subse­quently confirmed in both adults[6],[7],[8],[9],[10],[11],[12],[13] and chil­dren.[14],[15] More recently, "borderline" rejection (e.g. ai1at1) is included by some authors in the subclinical rejection category.[6],[8],[10],[11],[12] In a series of 330 consecutive protocol biopsies in patients on cyclosporine (CsA)-based therapy, and a prevalence of subclinical rejection of 15%, we found that 75% of subclinical rejection was Banff Type IA and 24 % was Type IB; arteritis (Banff IIA or greater) was found in a minority of cases.[16] More recently however, subclinical antibody-mediated rejection has been reported in ABO-incompatible transplants[16] and in trans­plant patients presensitized to donor HLA anti­gens.[17],[18]

Immunological Risk Factors for Subclinical Rejection

Subclinical rejection is associated with histo­incompatibility between the donor and reci­pient. In the Winnipeg studies, the prevalence of sub-clinical rejection in patients biopsied bet­ween months 1-3 was 0-25% with zero Class II mismatches; 30-32% with one class II mismatch; and 30-63% in patients with two Class II mis­matches.[19] A correlation between HLA mis­matches and the prevalence of subclinical re­jection has been reported also by others.[9],[13] Moreover, subclinical rejection is more preva­lent in patients presensitized to donor class I or class II HLA antigens.[20] Therefore, subclinical rejection appears to be an alloimmune response triggered by either mismatching of, or presensi­tization to the major histocompatibility antigens (see also previous Section).

Effect of the Immunosuppressive Regimen

The prevalence of subclinical rejection varies depending on whether antibody induction is used or not; and on the maintenance immuno­suppressive regimen. The first studies using pro­tocol biopsies were in patients that did not re­ceive antibody induction that were treated with CsA-based immunosuppressive regimens[5],[7],[8],[21],[22],[23] with[23] or without[5],[7],[8],[21],[22] MMF. The preva­lence of subclinical rejection between months one to three was approximately 25-30%. In some studies, the use of CsA microemulsion[24],[25] and MMF[13],[24],[25] reduced the prevalence of sub­clinical rejection; however in others it did not.[23] The introduction of tacrolimus (TAC) with[9],[10],[12],[24],[25] or without[5] MMF reduced the prevalence of subclinical rejection, compared to that observed with CsA. Jurewicz reported a prevalence of 18% of subclinical rejection (that included "bor­derline") at three months, in recipients of kid­neys from deceased donors treated with TAC, azathioprine and prednisone.[6] Nankivell et al reported subclinical rejection (including border­line) in over 50% at one month that was vir­tually abolished at three, six and 12 months in patients on TAC and MMF.[25] Gloor et al repor­ted a similar prevalence of subclinical rejection of only 2.6% at three months in patients on TAC and MMF; however more than 60% of patients received living donor grafts, and more than 50% received antibody induction.[10] In very early biopsies (mean time eight days post-trans­plant) in patients receiving TAC and steroids, plus MMF in two-thirds, Shapiro et al reported a 21% prevalence of borderline rejection and a prevalence of 25% of type I or type II rejection, despite stable or improving function.[9] We have recently reported a randomized, prospective, multicentre study that used TAC, MMF and prednisone as baseline immunosuppression in which the overall prevalence of subclinical re­jection between months one to six was only about 4%, although it ranged from 0% at 2 months to 8.9% at 6 months.[26]

The reports on early protocol biopsies using sirolimus (SRL) are few. In one study,[11] proto­col biopsies were performed at three months in patients randomized to SRL and low dose TAC ("CNI-sparing"), or SRL and MMF ("CNI-free"). Subclinical rejection was found in 6% and 15% of patients, and IF/TA was found in 53% and 15% of patients, in the CNI-sparing and CNI­free groups, respectively. In another study, 40 renal patients with HIV were treated with basi­liximab induction, SRL, CsA and prednisone. Protocol biopsies were obtained at one, six, 12 and 24 months post-transplant. The prevalence of subclinical rejection was 29% at the com­bined biopsy time points.[27] A more recent study has compared four different immunosuppressive regimens (without steroids) and monitored the patients with sequential protocol biopsies. In this study, 50 adult patients each were rando­mized to CsA/MMF, CsA/SRL, TAC/MMF and TAC/SRL. Protocol biopsies were done at one, two, six and 12 months and then yearly for five years. In the first year the prevalence of sub­clinical rejection was 22%, 8%, 16% and 6% respectively, in the order of immunosuppression regimens indicated above.[28]

The prevalence of subclinical rejection decrea­ses over time. In adults, after the first year, the overall prevalence of subclinical rejection was approximately 18%,[25] and at two years it was 8.9% in patients on TAC and 9.2% in patients on CsA.[29] In a follow-up of our prospective study of TAC and MMF-based immunosuppression, the prevalence of subclinical rejection at 24 months was only 1% (Rush D, Cockfield S, Nickerson P et al, Transplantation, in press). In children, the prevalence of subclinical rejec­tion also declined over time in one study, albeit less markedly. The prevalence of subclinical re­jection in one study was 50%, 32%, 19% and 16% at one, two, three and five years post­transplant, respectively.[14]

Significance of Subclinical Rejection in Protocol Biopsies

The treatment of subclinical rejection has been shown to reduce IF/TA[21] and improve graft function in two randomized studies in recipients of deceased[21] or living donor kidneys.[30] In both of the above studies, the immunosuppressive re­gimen included CsA. In the earlier study, the standard formulation CsA was used in combi­nation with azathioprine; whereas in the second study, CsA microemulsion or TAC, the latter in a small minority, was used in combination with MMF. The prevalence of subclinical rejection in the first three months post-transplantation was approximately 30% in the first study and approximately 15% in the second. In other stu­dies, the pathogenicity of subclinical rejection is suggested by the fact that IF/TA develops in patients in whom subclinical rejection is diag­nosed but not treated.[7],[8],[24],[31] These poor outcomes for undiagnosed or untreated subclinical rejec­tion have been reported for adult recipients of deceased donors.[7],[8],[21],[31] In a cohort of adult re­cipients of living donor grafts followed for 10 years, the diagnosis of subclinical rejection at 14 days post-transplant was associated with a significantly worse graft survival even if treated, perhaps because there was an increased inci­dence of later clinical rejections in such pa­tients.[13] In children, the finding of subclinical rejection in serial protocol biopsies (done at one, two, three and five years) was associated with progression of IF/TA and other changes of chronic injury as scored by the chronic allograft damage index (CADI),[32] as well as with dec­reased renal function and lower graft survival.[14]

The pathogenicity of sub-clinical rejection is supported also by the immunohistochemical cha­racterization of the graft-infiltrating cells and the analysis of the genes present in the graft across the range of acute inflammation. In the most recent and complete of such studies, Hoff­man et al concluded that subclinical rejection and clinical rejection are probably different sta­ges of the same process, as the differences in the immunophenotype of the infiltrating cells and the gene transcriptional findings observed between the two were quantitative more than qualitative. In clinical rejection, however, one novel finding was the increased expression of T-bet, a maturation factor for cytotoxic T cells.[33] Our group in Winnipeg has also studied the phenotypic and activation marker profile of graft-infiltrating cells by immunochemical me­thods[34] as well as the transcripts for a more li­mited number of pro-inflammatory and cytotoxic genes.[35] Our conclusions from these studies were essentially the same as those of Hoffman et al[33] namely, that subclinical and clinical rejection are likely different stages of the same poten­tially damaging alloimmune process. However, more recently, it has been suggested that pa­tients in whom the infiltrate in the protocol biopsy has a higher percentage of regulatory T­cells may have a better outcome than those in whom the infiltrate has fewer of these cells.[36]

Currently, and since our recent publication ci­ted previously,[26] we believe the prevalence of early subclinical rejection can be markedly re­duced in patients of low immunological risk given TAC and MMF, in whom the procure­ment of protocol biopsies for the sole purposes of detecting subclinical rejection may be there­fore unwarranted.

Peritubular Capillaritis

Although the Banff schema had a score for inflammation in the glomerular capillaries (glo­merulitis) since its first iteration, there had been no scoring of inflammation in the peritubular capillaries (PTC) until recently.[37] The associa­tion of PTC inflammation in early cellular or antibody-mediated rejection and the develop­ment of chronic rejection at later time points was first reported by Aita et al.[38] Moreover, Lerut et al demonstrated that PTC inflammation found in a protocol biopsy at three months post­transplant was associated with chronic rejection at one year.[39] In the most comprehensive study on the subject to date, Gibson et al[37] retrospec­tively analyzed 688 biopsies (46 preimplanta­tion, 461 protocol and 181 done for clinical indi­cations) and scored these as previously pro­posed in the Banff Conferences of 2003 and 2005. Peritubular capillaritis was found in 26.3% of biopsies overall, with a greater prevalence in clinically indicated biopsies (45.5%) as compared to protocol biopsies (17.6%). Of interest, how­ever, in the subgroup of patients with PTC in­flammation in protocol biopsies, those with di­ffuse capillaritis in at least one protocol biopsy had a significant decrease in glomerular filtration rate, as assessed by the Cockroft Gault equation, when compared to the group that never had PTC inflammation.[37]

Interstitial Fibrosis and Transplant Glomerulopathy

Interstitial Fibrosis

The more frequent sampling of the tubulo­interstitial space in small biopsy cores makes its scoring of IF/TA more useful as an outcome measure than the score of glomerular and vas­cular lesions. Moreover, the precise quantitation of the volume of interstitial fibrosis that can be obtained by histomorphometry,[40] and with the use of collagen-specific stains such as Sirius Red and image-analysis techniques,[41],[42] has been proposed as a more reliable index of tubulo-interstitial injury than that determined by the semi-quantitative scoring of the Banff[2] and CADI systems.[32]

Protocol biopsies obtained in the first year post-transplant have shown a rapid increase in the prevalence of IF/TA. Nankivell et al showed that, compared to biopsies performed at implan­tation or at one or two weeks, IF/TA increased by a factor of ten over the first post-transplant year, with less of an increase over the subse­quent nine years.[31] In a similar study of shorter duration with biopsies done at one, two, three and six months, our group reported negligible IF/TA at one and two months with a five-fold increase at six months.[22] The prevalence of IF/TA, a designation that is now preferred over the equally non-specific term CAN, in protocol biopsies at three to four months is between 24 and 42%,[7],[8],[43] at six months it is approximately 40%,[12],[44] and at one year it is approximately 50%.[14],[45] The prevalence of IF/TA is approxi­mately between 50% and 90% at two years.[7],[24],[29] All of the above studies were performed in patients under cyclosporine-based immunosu­ppression, except for the latter in which the presence of IF/TA was compared between CsA and TAC-treated patients, in both of whom the prevalence of IF/TA was approximately 70%.[29] In our study using tacrolimus and MMF, the prevalence of IF/TA (specifically ci + ct ≥2) in­creased from approximately 2.5% at implan­tation, to between 20 to 35% at six months, and to between 40 to 50% at 24 months (Rush D, Cockfield S, Nic-kerson P et al, Transplantation, in press). Finally, recent studies have reported the prevalence of IF/TA in protocol biopsies in patients on sirolimus.[28],[46],[47],[48],[49] In one study,[46] appro ximately 32% of patients switched to sirolimus at three months had new onset IF/TA at one year, as compared to 65% of those maintained on cyclosporine. In another,[47] patients randomized to sirolimus had a 34% prevalence of IF/TA at two years as compared with almost 80% for those on cyclosporine. In a third study, at five years IF/TA was present in 54% of CsA/MMF, 11.6% of CsA/ SRL, 38% of TAC/MMF and 14% of TAC/SRL-treated patients, respectively.[28] Finally, other studies have reported that patients randomized to sirolimus and maintained on that drug had a modest but significantly reduced IF/TA score as compared to patients rando­mized to and maintained on TAC[48] or CsA.[49] In most of the above studies the histopathological scoring was done according to the Banff schema.[2]

Other studies have used the CADI system for the scoring of chronic pathology.[50],[51] In the latter study,[51] protocol biopsies were obtained from patients in the US and Tricontinental Roche stu­dies at baseline, one and three years (n=739 for all biopsies). In these patients that were treated with CsA, MMF and prednisone, the mean CADI score more than doubled between base­line and 12 months and more than tripled by 36 months.

Risk Factors for Interstitial Fibrosis and Tubular Atrophy

By multivariate analysis, a number of risk fac­tors have been correlated with the development of IF/TA that occurs in the first six to 12 months post-transplant. These include donor age, recipient gender (female), ischemia-reperfusion, subclinical and clinical rejections, delayed graft function, and nephrocalcinosis related to hyper­parathyroidism.[8],[21],[26],[44],[51] Fibrosis beyond one year has been correlated with donor age, subclinical and clinical rejections, infection with cytome­galovirus, and episodes of presumed cyclos­porine nephrotoxicity in some studies,[14],[24],[29],[46],[47],[48],[49] but not others.[51] The data on sirolimus that were obtained in randomized studies[28],[46],[47],[48],[49] (see pre­vious section) although preliminary, are intri­guing, and suggest that a maintenance drug regimen that includes a calcineurin inhibitor (CNI) may be a risk factor for late onset in­terstitial fibrosis. In our study using tacrolimus and MMF, the only risk factor for IF/TA (ci + ct ≥2) at 24 months was having been the reci­pient of a kidney from a deceased donor; whereas there was significantly less IF/TA in those patients whose hypertension was treated with angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers than in those in whom other types of antihypertensives were used (Rush D, Cockfield S, Nickerson P et al, Transplantation, in press).

Significance of IF/TA in Protocol Biopsies

The detection of IF/TA in protocol biopsies procured as early as three to six months post­transplant in well functioning transplants has been correlated with later allograft dysfunction and loss.[8],[22],[31],[52] Similarly, increased chronic scores reported with the CADI system[32] at one year[51] or two years,[50] have been correlated with graft losses at three years and six years, respec­tively. Furthermore, the more precise measure­ment of interstitial fibrosis at six months using morphometry[40] or Sirius Red staining[41] has been correlated with graft survival and time to graft failure, respectively. It has now become apparent that patients with concomitant interstitial infla­mmation and fibrosis may have a greater risk of graft dysfunction and loss than those patients with fibrosis alone, as has been reported in adults[22],[41],[53],[54] and in children.[14] The degree of inflammation may be irrelevant -i.e. it may be below the Banff threshold for borderline rejec­tion.[53] Indeed, sequential protocol biopsies that show persistent inflammation below the Banff threshold for rejection may be associated with decreased renal function after one or two years.[55]

Transplant Glomerulopathy

The early histological and clinical descriptions of transplant glomerulopathy (TG) in adults[56],[57] and children[58] refer to its usual presentation late post-transplantation, although earlier presenta­tions were also recognized,[57],[58] typically with graft dysfunction, proteinuria and a poor prog­nosis. With the use of protocol biopsies, the Mayo Clinic group at Rochester has contributed greatly to the clinical and pathological under­ standing of this entity.[53],[59]

The cumulative incidence of TG increases from 4% at one year to approximately 20% at five years. In 50% of cases the diagnosis is subclin­ical -i.e. it is found on protocol biopsy; most of these in the first 15 months post-transplant. The early diagnosis of TG may be made in patients that do not have significant proteinuria or impaired renal function, and TG may exist in the absence of other histological changes such as IF/TA.[59] The prognosis is the worst of all pathologies diagnosed on protocol biopsy.[53] Risk factors for TG detected on protocol biopsy are prior rejection and presensitization to class II HLA antigens.[59]

In conclusion, immunological risk factors are undoubtedly important in determining graft sur­vival, as demonstrated by the correlation between graft survival and HLA matching.[60] Moreover, a recent study has shown that most graft losses are due to alloreactivity, particularly alloantibody against donor HLA.[61] Protocol biopsies have shown that much of this alloreactivity, at least early on is subclinical.

The optimal use of immunosuppressive agents requires that the anti-donor alloimmune res­ponse be monitored by non-invasive tests that would allow for frequent determinations of that response. The urine may be the best medium to look for candidate tests and our group is exa­mining the use of specific chemokines,[62] as well as interrogating the urine proteome[63] and meta­bolome[64] in an attempt to develop such a test.